Fecal streptococci speciating media

ABSTRACT

A media to simplify the speciation of fecal streptococci. The media is a gelatin with additives of peptone, esculin, ferric ammonium citrate and sodium azide in a basic form and may include sodium citrate in a modified form thereof.

United States Patent [191 Galvani FECAL STREPTOCOCCI SPECIATING MEDIA Inventor: Mary M. Galvani, 540 Mississippi River Blvd, St. Paul, Minn. 551 16 Filed: July 18, 1973 Appl. No.: 380,141

Published under the Trial Voluntary Protest Program on January 28, 1975 as document no. B 380,141.

[1.8. CI. 195/100; 195/1035 R Int. Cl. C12K 1/10 Field of Search 195/99, 100, 101, I02.

[ Dec. 9, 1975 {56] References Cited OTHER PUBLICATIONS Difio Manual, pp. 46-52, 9th Edition, 1953.

Primary ExaminerA. Louis Monacell Assistant Examiner-Robert J. Warden Attorney, Agent, or FirmJames R. Cwayna 8 Claims, No Drawings FECAL STREPTOCOCCI SPECIATING MEDIA The need for clean, usable waters is known to virtually all of the persons of the world. The only hope to provide such clean, useable waters lies in the detection of the contaminants, the location of the source of discharge of these contaminants and the control of such discharge. Unless there is a simple means provided which will permit the accurate analyzing of fecal contamination and the verification of its source there will be no solution to this problem. Through the speciation of streptococci found in any body of water, the exact origin of fecal contamination can be determined and more importantly, the source of discharge can be located by the increase in magnitude of the number of streptococci present in selected geographical samplings.

The problem and thus the solution lies in providing an accurate, simple method to make this speciation.

Presently, total coliform, and fecal coliform in larger laboratories, is used as a baseline indicator system for evaluating the microbiological suitability of drinking and recreational waters. Coliform is not a reliable index for two reasons. First, authorities agree that the procedure used in analyzing colifonn Most Probable Number" is not accurate. Secondly, fecal coliform is too thermo-sensitive to be used in small laboratories and speciation of fecal coliform required to indicate the source of origin of the fecal matter is so complicated that it is only a research problem.

The preferable indicator system is fecal streptococci because they are not thermo-sensitive, they readily lend themselves to effective speciation and definite key streptococci, through their biochemical reaction, manifest the source of the fecal origin. The reluctance to use streptococci in the past as an indicator of fecal contamination was based on the fact that in the analysis of fecal strectococci, a strain of strectococci predominated. These were Streptococcifaecalis variations liquefaciens (S. faecalis var. liquefaciens). These liquefaciens were reported to be ubiquitious, present in everything, and not peculiar to fecal contamination. So that in studying fecal contamination via fecal streptococci the analyst could not tell without further speciation whether or not it was the streptococci liquefaciens that purportedly do not mean anything. Speciation required expert technique, weeks of subculturing and years of study.

The media which is the subject of this application requires only one simple subculture followed by incubation in a refrigerator and then in the incubator to: positively identify S. Liquefaciens, to separate the streptococci peculiar to animal from those common to both man and animal; and to purify the streptococci inoculum so that it can be easily subcultured to positively identify its species. The response of the media to growth of bacteria is dramatic and easily read.

It is therefore an object of applicants invention to provide a simple means for the detection of the presence of human sewage in waters and other media.

it is further object of applicants invention to provide a simple media for evaluating the microbiological suitability of drinking and recreational waters.

It is a further object of applicants invention to provide a simple media to measure the efficiency of sewage treatment plants both at discharge of effluent and down stream.

it is a further object of applicants invention to provide a simple media to ultimately determine the species of streptococci.

It is a further object of applicants invention to provide a single subculture media to follow a streptococci count made on M.Enteroc0ccus agar which simultaneously identifies the presence of Streptococci faecalis variation liquefaciens (S. faecalis var. liquefaciens) or Streptococcifaecalis variation zymogens (S. faecalis var. zymogens) and which requires only one additional subculture to differentiate between the two variations.

It is still a further object of applicants invention to provide a media which will simply and positively identify streptococci that are peculiar to animal, these being Streptococci bovis and Streptococci equinus.

It is still a further object of applicants invention to provide a media that sustains and nourishes Streptococci inhibiting contaminants transferred during innoculation thereby rendering it suitable as a basic culture for further subcultures.

It is another object of the invention to enable the analyst to correlate the colony morphology of the 48 hour M.Enter0coccus with the easy speciation thus evantually enabeling the user to judge species even without speciation.

It is another object of the invention to provide a media which can be easily checked to determine whether the inoculum is a mixed culture.

These and other objects and advantages of applicants invention will more fully appear from a consideration of the accompanying disclosure and claims.

in order to provide applicants fecal streptococci speciating media, the following formulation is utilized; and the amounts for each ingredient are expressed in grams per liter.

Peptone 10.0-30.0 Esculin 0. l05 .00 Ferric Amonium Citrate 0. |0-3.00 Sodium Azide 0.0l-0.S0 Bacteriological Gelatin S0.0200.00

In the basic formulation set forth above, it is essential that the bacteriological gelatin be maintained at a pH level of 6.8.

In the basic formulation, applicant has found that Sodium Citrate in the range of 0.1 to 3.0 grams/liter may be beneficially added but is not totally essential to the performance of the media.

Within this formulation, applicant has found that the most desirable results have been accomplished with a formulation as follows:

Peptone 20.0 Esculin 1.0 Ferric Ammonium Citrate 0.5 Sodium Azide 0.4 Bacteriological Gelatin 1200 If the addition of Sodium Citrate is desired in this specific formulation then 1.0 gram/liter should be added.

With this more specific formulation, the same conditions with regard to pH level must be maintained and the amounts have again been express" in grams/liter.

The preparation of the media rfiiiires a minimal amount of equipment and skill. the dry ingredients are finelly powdered and mraughly mixed; 143,00

grams of the mixture are placed in a beaker and cold distilled water is added gradually until the media is throughly wetted and evenly suspended. This mixture is then slowly heated to boiling with constatn vigorous agitation until the media boils in large bubbles. At this point, the media is removed from heat and allowed to cool. Three milliliters (3ml.) aliquots are dispersed in small tubes and capped. The media is then autoclaved for ID minutes at ten pounds pressure at 116 Centigrade or 240 Fahrenheit; the pH of the media is 6.8.

The media may be provided in powdered, dehydrated from. The media may be provided in prepared form in tubes containing 3ml., this may be rejuvinated, if required after long standing, by placing the same in a waterbath maintained at 50 Centigrade for a period of minutes and then refrigerated until the media is in a solid form.

The use of the media is also simple and requires a minimal amount of either equipment or skill.

Red and pink colonies appear on a 48-hour culture of M.Enter0coccus. These colonies are picked one by one with an inocculating needle and plunged into the previously refrigerated streptococci speciating media, one colony being introduced to each tube.

A rack of these tubes is placed under refrigeration at 9.5 Centrigrade, plus or minus 0.5 Centrigrade. Growth is indicated by the appearance of a black precipitate in the clear media. Animal streptococci, being S .Bovis and S .Equinus will show no growth. SJiquefaciens grow easily, showing precipitate all along the innoculation stab. SJ'uecuIis and the other streptococci common to both man and animal show growth.

After 4 days, the rack of tubes is placed in an incubator at 355 plus or minus 0.5 Centigrade. Growth is then demonstrated by all tubes turning completely black in from 8 to 24 hours. After 48 hours of incubation, the rack of tubes is taken out of the incubator and placed in the refrigerator for 30 minutes. The media in all tubes becomes solid except those that contain Sliquefaciens, which remains liquid.

The new media presented herein greatly simplifies the speciation of streptococcus faecalis var. liquefaciens which has been labeled ubiquitous and has been tagged as having limited sanitary significance.

The procedure with the new media requires less understanding for accurate results than previous techniques. It may be performed in the poorest of equipped laboratories while affording excellent results.

It should be obvious then that applicant has provided a new and unique media for fecal streplococci speciation that will provide accurate analysis and provide such analysis within a short period of time.

What I claim is:

l. A fecal streptococci speciating media consisting of a mixture of Peptone, Esculin, Ferric Ammonium Citrate Sodium Azide and Bacteriological Gelatin having a pH of 6.8.

2. The media set forth in claim 1 and including Sodium Citrate.

3. The media set forth in claim 1 and the amounts of the said ingredients being in the ranges of: Peptone, 10.0 to 30.0 grams per liter; Esculin, 0.10 to 5.00 grams per liter; Ferric Ammonium Citrate, 0.10 to 3.00 grams per liter; Sodium Azide, 0.01 to 0.50 grams per liter; and Bacteriological Gelatin, 50.0 to 200.00 grams per liter.

4. The media set forth in claim 3 and Sodium Citrate in the range of 0.1 to 3.0 grams per liter.

5. The media set forth in claim 1 and the amounts of said ingredients being: Peptone, 20.0 grams per liter; Esculin, 1.0 grams per liter; Ferric Ammonium Citrate, 0.5 grams per liter; Sodium Azide, 0.4 grams per liter; and Bacteriological Gelatin, 120.0 grams per liter.

6. The media set forth in claim 5 and including Sodium Citrate in the amount of 1.0 grams per liter.

7. The process and method of providing a fecal streptococci speciating media including the steps of providing a selection of elements including:

a. Peptone, Esculin, Ferric Ammonium Citrate, So-

dium Azide and Bacteriological Gelatin;

b. providing all the dry ingredients in powdered form and thoroughly mixing the same;

c. providing 143.00 grams of the dry, mixed ingredients for mixture in cold distilled water to provide a thoroughly wetted and evenly suspended mixture;

d. slowly heating the mixture to boiling while constantly agitating the same;

e. cooling the mixture immediately after reaching the boiling point;

f. autoclaving the resulting mixture as selected; and

g. adjusting the pH of the mixture to 6.8.

8. The process and method of claim 7 wherein the elements include Sodium Citrate. 

1. A FECAL STREPTOCOCCI SPECIATING MEDIA CONSISTING OF A MIXTURE OF PEPTONE, ESCULIN, FERRIC AMMONIUM CITRATE SODIUM AZIDE AND BACTERIOLOGICAL GELATIN HAVING A PH OF 6.8.
 2. THE MEDIA SET FORTH IN CLAIM 1 AND INCLUDING SODIUM CITRATE.
 3. THE MEDIA SET FORTH IN CLAIM 1 AND THE AMOUNTS OF THE SAID INGREDIENTS BEING IN THE RANGES OF: PEPTONE, 10.0 TO 30.0 GRAMS PER LITER; ESCULIN, 0.10 TO 5.00 GRAMS PER LITER; FERIC AMMONIUM CITRATE, 0.10 TO 3.00 GRAMS PER LITER; SODIUM AZIDE. 0.01 TO 0.50 GRAMS PER LITER; AND BACTERIOLOGICAL GELATIN, 50.0 TO 200.00 GRAMS PER LITER.
 4. The media set forth in claim 3 and Sodium Citrate in the range of 0.1 to 3.0 grams per liter.
 5. The media set forth in claim 1 and the amounts of said ingredients being: Peptone, 20.0 grams per liter; Esculin, 1.0 grams per liter; Ferric Ammonium Citrate, 0.5 grams per liter; Sodium Azide, 0.4 grams per liter; and Bacteriological Gelatin, 120.0 grams per liter.
 6. The media set forth in claim 5 and including Sodium Citrate in the amount of 1.0 grams per liter.
 7. THE PROCESS AND METHOD OF PRPROVIDING A FECAL STREPTOCOCCI SPECIATING MEDIA INCLUDING THE STEPS OF PROVIDING A SELECTION OF ELEMENTS INCLUDING: A. PEPTONE, ESCULIN, FERRIC AMMONIUM CITRATE, SODIUM AZIDE AND BACTERIOLOGICAL GELATIN; B. PROVIDING ALL TH DRY INGREDIENTS IN POWDERED FORMAND THOROUGHLY MIXING THE SAME; C. PROVIDING 143.00 GRAMS OF THE DRY, MIXED INGREDIENTS FOR MIXTURE IN COLD DISTILLED WATER TO PROVIDE A THOROUGHLY WETTED AND EVENLY SUSPENDED MIXTURE; D. SLOWLY HEATING THE MIXTURE TO BOILING WHILE CONSTNTLY AGITATING THE SAME; E. COOLING THE MIXTURE IMMEDIATELY AFTER REACHING THE BOILING POINT; F. AUTOCLAVING THE RESUKULTING MIXTURE AS SELECTED; AND G. ADJUSTING THE PH OF THE MIXTURE TO 6.8.
 8. The process and method of claim 7 wherein the elements include Sodium Citrate. 